Method for finger-printing heparins

Abstract

Methods to generate a distinctive fingerprint (pattern of migration) for a sample of complex, polydisperse heparins are provided. The methods involve adding resolving agents such as polyamines to a heparin sample and then analyzing the sample with a technique that separates macromolecules according to charge to mass ratio (e.g. capillary electrophoresis). The resulting electropherogram is unique to and characteristic of the heparin sample. The methods may be used, for example, to monitor the quality and consistency of various heparin preparations.

Description

BACKGROUND OF THE INVENTION[0001]1. Field of the Invention[0002]The invention generally relates to methods to “fingerprint” or resolve the migration patterns of complex, polydisperse heparin mixtures. In particular, the invention provides methods for resolving complex heparins by adding polyamine resolving agents to the mixture prior to analysis of the heparins by a technique that separates macromolecules according to charge to mass ratio.[0003]2. Background of the Invention[0004]Anticoagulants are molecules used to treat and prevent a number of thrombotic disorders including pulmonary embolism, deep-vein thrombosis, disseminated intravascular coagulation, acute myocardial infarction, unstable angina, cerebrovascular thrombosis, and others. Nearly 576,000 new cases of deep vein thrombosis and pulmonary embolism, two of the most common thrombotic conditions, are diagnosed every year in the US (1). Thus, a huge market exists for anticoagulants.[0005]The two most commonly used anticoag...

AI Technical Summary

Benefits of technology

[0011]A method has been developed to accurately and consistently fingerprint unfractionated, polydisperse heparin mixtures. The method resolves broad heterogeneous heparin peaks into numerous (>20) components in a reliable, highly reproducible manner. This high level of resolution is attained by adding to the heparin sample polyamine resolving agents. When the heparin / polyamine mixture is analyzed using, for example, capillary electrophoresis (CE), the resulting electropherogram constitutes a high-resolution “fingerprint” that is characteristic of the heparin sample. Comparison of the fingerprints from two different heparin samples can reveal whether the two samples are the same or different. Thus, the method can be used, for example, to characterize heparin samples of known composition (e.g. to establish a reference or control sample), and to identify unknown samples by comparison to the reference. The method is therefore useful, for example, to monitor product quality during various stages of preparation, to monitor batch to batch variations, and to authenticate samples of which the composition or origin may be in doubt. Because the method is very fast, it is amenable to inclusion in “in-line” analyses such as those used during the preparation of heparin products, or for the consecutive analyses of large numbers of heparin samples on an industrial scale.

Problems solved by technology

Yet, they suffer from significant problems including enhanced bleeding risk, immunological reaction, patient-to-patient response variability, narrow therapeutic index, poor oral bioavailability, the need for frequent coagulation monitoring, and high cost to benefit ratio.

The many adverse side effects of heparin and LMWH arise from their structure.

Yet, these interactions are different and unpredictable for different heparin or LMWH chains because their microscopic structures are different.

These differences are perhaps the single major source of complications associated with heparin therapy.

Thus, patients on enoxaparin may not be routinely switched to tinzaparin and vice versa.

Both these techniques resolve UFH and LMWH into oligomers, especially the smaller chains, but do not provide more detailed structural information.

Unfortunately, these powerful systems work primarily on fragmented or smaller heparin oligomers.

Analysis of unfragmented, intact LMWHs and unfractionated heparin is challenging because of the size of the biopolymers as well as its phenomenal charge.

Typically, the main disadvantage of these polymeric species is that a wide peak is generally observed.

Yet, the resolution of LMWH samples was minimal.

These approaches are not useful to perform analysis of clinical LMWHs for assessing product identity and product quality.

Thus, although several analytical methods are available, the prior art has thus far failed to provide an analytical method that can be implemented in routine manner to assess product identity and quality, and to authenticate the purported composition of heparin samples.

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